This protocol was used to clean up DNA extracted from plasmids which contained impurities which subsequently interfered with restriction enzyme digestion.
The volume of the solution containing 10-250 fig of DNA was adjusted to 250 fi\
with dHgO, and 15 fii of ethidium bromide (10 mg/ml) and 140 /xl of 7.5 M ammonium acetate were added and the contents of the tube mixed by vortexing. To this solution, 420 /xl of a 5:1 mixture of phenol:chloroform was added, mixed well and spun at 13,000 rpm using a microfuge for 2 min. The aqueous phase was carefully removed and two volumes of 100 % ethanol were added and the tube was incubated at room temperature for 2 min and then spun at 13,(XX) rpm for 5 min to pellet the DNA. The pellet was washed in cold 70 % ethanol, dried in a speedvac and redissolved in TE as previously described.
2.8.3 Extraction of messenger RNA
Extraction of mRNA from adult rat kidney and whole Wistar rat embryos was carried out using a commercial kit (Micro-Fast Track™ mRNA Isolation Kit, Version 2.0, Invitrogen*, USA). The kit recommended starting with 10-2(X) mg of tissue per isolation. Six randomly selected embryos at E8, E9, ElO, E ll and one E14 embryo were dissected from the uterine horns of timed-mated Wistar rats (Charles River Ltd., UK) in sterile PBS, rapidly transferred into sterile 1.5 ml Eppendorfs and snap frozen in liquid nitrogen (BOC, UK).
Embryos were randomly selected from two pooled litters, after any embryos which were obviously too young, old or deformed had been removed. A kidney from one of the mothers was also removed and all fat and encapsulating connective tissue cut away. The kidney was sliced up, and a piece weighing approximately 10 mg was snap frozen as described above.
Any components provided with commercial kits described henceforth are italicised. The Lysis Buffer was prepared by adding 20 /xl of Protein/RNase Degrader
(proprietary) to 1 ml of Stock Buffer per tissue sample. The components of the Stock
Buffer were:- 200 mM NaCl; 2(X) mM Tris pH 7.5; 1.5 mM MgCl2‘, 2 % SDS. The Lysis
Buffer was pre-heated in a water bath to 45°C, and 1 ml was added to each tube. The
tissue was then homogenized by repeatedly drawing up and expelling through an 18-21 gauge sterile needle (Becton Dickinson, Ireland) attached to a 1 ml sterile syringe (Becton Dickinson, Ireland).
The homogenates were then incubated for 20 min in a slow-shaking water bath at 45°C. After this incubation some insoluble material was evident in the E14 and kidney preparations and the tubes were spun for 5 min at 13,000 rpm at room temperature using
a microfuge to pellet this material. The supernatants were transferred to a fresh tube. The NaCl concentration of the lysates were adjusted to 0.5 M by the addition of 63 /xl of 5 M NaCl stock solution (supplied with the kit). The lysates were then mixed thoroughly and the DNA sheared by repeatedly drawing up and expelling through an 18- 21 gauge sterile needle as before.
One Oligo (dT)2o.3o Cellulose Tablet was added to each lysate, the tube lids were
closed and the tablets allowed to swell and break up for 2 min. The tubes were then gently shaken at room temperature for 20 min.
The oligo (dT) cellulose was then pelleted by spinning for 8 min at 13,000 rpm using a microfuge and the supernatant gently taken off. The oligo (dT) cellulose was then resuspended in 1.3 ml of Binding Buffer. The components of Binding Buffer were:-500 mM NaCl; 10 mM Tris-HCl, pH 7.5 (made up in DEPC-treated water). The oligo (dT) cellulose was then pelleted by spinning for 5 min at 13,000 rpm using a microfuge and the supernatant gently taken off. This process was repeated twice more and then the oligo (dT) cellulose was resuspended in 300 /xl of Binding Buffer.
The samples were then pipetted into spin columns (supplied with the kit) which were then placed into sterile RNase-free microfuge tubes (supplied with the kit) and spun for 10 sec at 13,000 rpm to transfer the cellulose into the spin column. This step was repeated. The eluant in the tube was discarded and the columns washed by filling the spin column to the brim with Binding Buffer and spinning the tubes for 10 sec. This wash step was repeated three more times.
The non-polyadenylated RNA was removed by the addition of 200 /xl of Low Salt
Wash Buffer. This was gently mixed into the cellulose bed using a sterile pipette tip. The
DEPC-treated water. The tubes were spun for 10 sec at 13,000 rpm, and this step was repeated once.
The spin column were then transferred into fresh tubes and 100 /d of Elution
Buffer was mixed into the bed and the tubes spun for 10 sec at 13,000 rpm. The eluant
was retained in the tube and a second 100 /d of Elution Buffer was mixed into the bed and spun. The components of the Elution Buffer were:- 10 mM Tris-HCl pH 7.5 made up in DEPC-treated water. If there was less than 200 /d of eluant in the bottom of the tube the tubes were spun for 1 min at 13,000 rpm. The spin columns were then discarded.
The polyadenylated RNA was precipitated by the addition of 10 /xl of glycogen (supplied as a 2 mg/ml stock made up in DEPC-treated water), 30 /xl of 2 M sodium acetate (supplied in the kit) and 600 /xl of 100 % ethanol. The mRNA was allowed to precipitate overnight at -20°C, then spun at 13,(XX) rpm at 4°C for 15 min, the ethanol was removed and the pellets washed in ice-cold 70 % ethanol. Finally the pellets were dried using a Speedvac, and resuspended in 10 /xl of DEPC-treated water.